In silico analysis of the substitution mutations and evolutionary trends of the SARS-CoV-2 structural proteins in Asia.

Objectives: To address a highly mutable pathogen, mutations must be evaluated. SARS-CoV-2 involves changing infectivity, mortality, and treatment and vaccination susceptibility resulting from mutations. Materials and Methods: We investigated the Asian and worldwide samples of amino-acid sequences (AASs) for envelope (E), membrane (M), nucleocapsid (N), and spike (S) proteins from the announcement of the new coronavirus 2019 (COVID-19) up to January 2022. Sequence alignment to the Wuhan-2019 virus permits tracking mutations in Asian and global samples. Furthermore, we explored the evolutionary tendencies of structural protein mutations and compared the results between Asia and the globe. Results: The mutation analyses indicated that 5.81%, 70.63%, 26.59%, and 3.36% of Asian S, E, M, and N samples did not display any mutation. Additionally, the most relative mutations among the S, E, M, and N AASs occurred in the regions of 508 to 635 AA, 7 to 14 AA, 66 to 88 AA, and 164 to 205 AA in both Asian and total samples. D614G, T9I, I82T, and R203M were inferred as the most frequent mutations in S, E, M, and N AASs. Timeline research showed that substitution mutation in the location of 614 among Asian and total S AASs was detected from January 2020. Conclusion: N protein was the most non-conserved protein, and the most prevalent mutations in S, E, M, and N AASs were D614G, T9I, I82T, and R203M. Screening structural protein mutations is a robust approach for developing drugs, vaccines, and more specific diagnostic tools.

The origin and evolution of emerged swine acute diarrhea syndrome coronavirus with zoonotic potential.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered alphacoronavirus with zoonotic potential that causes diarrhea and vomiting mainly in piglets. Having emerged suddenly in 2017, the prevailing opinion is that the virus originated from HKU2, an alphacoronavirus whose primary host is bats, and at some unknown point achieved interspecies transmission via some intermediate. Here, we further explore the evolutionary history and possible cross-species transmission event for SADS-CoV. Coevolutionary analysis demonstrated that HKU2 may have achieved host switch via SADS-related (SADSr)-CoV, which was isolated from the genus Rhinolophus in 2017. SADS-CoV, HKU2, and SADSr-CoV share similar codon usage patterns and showed a lower tendency to use CpG, which may reflect a method of immune escape. The analyses of virus-host coevolution and recombination support SADSr-CoV is the direct source of SADS-CoV that may have undergone recombination events during its formation. Structure-based spike glycoprotein variance analysis revealed a more nuanced evolutionary pathway to receptor recognition for host switch. We did not find a possible positive selection site, and the dN/dS of the S gene was only 0.29, which indicates that the current SADS-CoV is slowly evolving. These results provide new insights that may help predict future cross-species transmission, and possibly surveil future zoonotic outbreaks and associated public health emergencies.

A new PEDV strain CH/HLJJS/2022 can challenge current detection methods and vaccines.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) variant strains cause great economic losses to the global swine industry. However, vaccines do not provide sufficient protection against currently circulating strains due to viral mutations. This study traced the molecular characteristics of the most recent isolates in China and aimed to provide a basis for the prevention and treatment of PEDV. METHODS: We obtained samples from a Chinese diarrheal swine farm in 2022. Reverse transcription polymerase chain reaction and immunofluorescence were used to determine the etiology, and the full-length PEDV genome was sequenced. Nucleotide similarity was calculated using MEGA to construct a phylogenetic tree and DNASTAR. Mutant amino acids were aligned using DNAMAN and modeled by SWISS-MODEL, Phyre2 and FirstGlance in JMOL for protein tertiary structure simulation. Additionally, TMHMM was used for protein function prediction. RESULTS: A PEDV virulent strain CH/HLJJS/2022 was successfully isolated in China. A genome-wide based phylogenetic analysis suggests that it belongs to the GII subtype, and 96.1-98.9% homology existed in the whole genomes of other strains. For the first time, simultaneous mutations of four amino acids were found in the highly conserved membrane (M) and nucleocapsid (N) proteins, as well as eight amino acid mutations that differed from the vast majority of strains in the spike (S) protein. Three of the mutations alter the S-protein spatial structure. In addition, typing markers exist during strain evolution, but isolates are using the fusion of specific amino acids from multiple variant strains to add additional features, as also demonstrated by protein alignments and 3D models of numerous subtype strains. CONCLUSION: The newly isolated prevalent strain CH/HLJJS/2022 belonged to the GII subtype, and thirteen mutations different from other strains were found, including mutations in the highly conserved m and N proteins, and in the S1° and COE neutralizing epitopes of the S protein. PEDV is breaking through original cognitions and moving on a more complex path. Surveillance for PEDV now and in the future and improvements derived from mutant strain vaccines are highly warranted.

Evolutionary analysis of Omicron variant BF.7 and BA.5.2 pandemic in China.

On December 7, 2022, China adjusted public health control measures, there have been widespread of SARS-CoV-2 infections in Chinese mainland. As the number of infected people increased, the mutation probability of SARS-CoV-2 is also raised. Therefore, it is of great importance to monitor SARS-CoV-2 variants and its mutations in China. In this current study, 665 SARS-CoV-2 genomes from China deposited in the public database were used to analyze the proportion of different variants; to determine the composition of variants in China across different provinces; and analyze specific mutation frequency, focusing on 12 immune escape residues. The results showed that no new mutations were generated on the 12 immune escape residues. The evolutionary analysis of the BF.7 variant circulating in China showed that there is an independent evolutionary branch with unique mutation sites, officially named BF.7.14 by PANGO. This variant may have been imported from Russia to Inner Mongolia at the end of September 2022 and continued its spread in China. The evolutionary analysis of BA.5.2 variant shows that the variant is composed of two sub-variants, named BA.5.2.48 and BA.5.2.49 by PANGO, respectively. This variant may have been imported from abroad to Beijing at the beginning of September 2022 and formed two sub-variants after domestic transmission. Finally, this study showed that current epidemic variants in China were already circulating in other countries, and there were no additional mutations on immune escape residues that could pose a threat to other countries.

Mutations in SARS-CoV-2 structural proteins: a global analysis.

BACKGROUND: Emergence of new variants mainly variants of concerns (VOC) is caused by mutations in main structural proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, we aimed to investigate the mutations among structural proteins of SARS-CoV-2 globally. METHODS: We analyzed samples of amino-acid sequences (AASs) for envelope (E), membrane (M), nucleocapsid (N), and spike (S) proteins from the declaration of the coronavirus 2019 (COVID-19) as pandemic to January 2022. The presence and location of mutations were then investigated by aligning the sequences to the reference sequence and categorizing them based on frequency and continent. Finally, the related human genes with the viral structural genes were discovered, and their interactions were reported. RESULTS: The results indicated that the most relative mutations among the E, M, N, and S AASs occurred in the regions of 7 to 14, 66 to 88, 164 to 205, and 508 to 635 AAs, respectively. The most frequent mutations in E, M, N, and S proteins were T9I, I82T, R203M/R203K, and D614G. D614G was the most frequent mutation in all six geographical areas. Following D614G, L18F, A222V, E484K, and N501Y, respectively, were ranked as the most frequent mutations in S protein globally. Besides, A-kinase Anchoring Protein 8 Like (AKAP8L) was shown as the linkage unit between M, E, and E cluster genes. CONCLUSION: Screening the structural protein mutations can help scientists introduce better drug and vaccine development strategies.

Bioinformatics analysis on structure and function and expression vector construction of SARS-CoV-2 related protein TMPRSS2

Aim Human TMPRSS2 is a transmembrane serine protease.In this paper, the structure and function of the protein were systematically analyzed by bioinformatics, the codon was optimized and the pro- karvotie expression vector was constructed to explore the molecular mechanism of SARS-CoV-2 infecting host cells.Methods The recombinant expression vector pET-22b-TMPRSS2 was generated by molecular cloning technology.The homology, functional sites, subcellular localization, three-dimensional structure and evolutionary characteristics of TMPRSS2 protein were systematically analyzed by using analytical tools such as Protparam, NetPhos3.1, Blast, Clustal X2 and MEGA7.0.Results The prokarvotic expression plas- mid was constructed correctly;TMPRSS2 belongs to medium molecular weight protein, which is composed of 492 amino acid residues.The theoretical isoelectric point is 8.12, the molecular extinction coefficient is 118 145 L * mol~1 * cm"1 , and the half-life is 30 h;TMPRSS2 has 15 potential glycosylation sites and 49 possible phosphorylation sites.It is a transmembrane hydrophilie protein without signal sequenee.In addition, the protein has 13 potential B-cell epitopes and 7 T-eell epitopes.Seeondarv structure analysis showed that random coil accounted for the highest proportion of TMPRSS2 protein ( 0.453 3) , followed by extended strand (0.252 0).Sequence comparison and evolutionary analysis showed that the highest sequence consistency and closest genetic relationship with human TMPRSS2 was Pan troglodytes, followed by gorilla.Conclusions Human-derived TMPRSS2 protein is ev- olutionarilv conserved and functionally important.Hie results of this study can help to reveal the structure and mechanism of action of TMPRSS2 protein, provide ideas for the diagnosis and treatment of COYID-19, and accelerate the research and development process of new drugs targeting TMPRSS2 protein. Copyright © 2022 Publication Centre of Anhui Medical University. All rights reserved.

Designing potential siRNA molecules for silencing the gene of the nucleocapsid protein of Nipah virus: A computational investigation.

Nipah virus (NiV), a zoonotic virus, engenders severe infections with noticeable complications and deaths in humans and animals. Since its emergence, it is frightening, this virus has been causing regular outbreaks in various countries, particularly in Bangladesh, India, and Malaysia. Unfortunately, no efficient vaccine or drug is available now to combat this baneful virus. NiV employs its nucleocapsid protein for genetic material packaging, which is crucial for viral replication inside the host cells. The small interfering RNAs (siRNAs) can play a central role in inhibiting the expression of disease-causing viral genes by hybridization and subsequent inactivation of the complementary target viral mRNAs through the RNA interference (RNAi) pathway. Therefore, potential siRNAs as molecular therapeutics against the nucleocapsid protein gene of NiV were designed in this study. First, ten prospective siRNAs were identified using the conserved nucleocapsid gene sequences among all available NiV strains collected from various countries. After that, off-target binding, GC (guanine-cytosine) content, secondary structure, binding affinity with the target, melting temperature, efficacy analysis, and binding capacity with the human argonaute protein 2 (AGO2) of these siRNAs were evaluated to predict their suitability. These designed siRNA molecules bear promise in silencing the NiV gene encoding the nucleocapsid protein and thus can alleviate the severity of this dangerous virus. Further in vivo experiments are recommended before using these designed siRNAs as alternative and effective molecular therapeutic agents against NiV.

Application of Bayesian phylogenetic inference modelling for evolutionary genetic analysis and dynamic changes in 2019-nCoV.

The novel coronavirus (2019-nCoV) has recently caused a large-scale outbreak of viral pneumonia both in China and worldwide. In this study, we obtained the entire genome sequence of 777 new coronavirus strains as of 29 February 2020 from a public gene bank. Bioinformatics analysis of these strains indicated that the mutation rate of these new coronaviruses is not high at present, similar to the mutation rate of the severe acute respiratory syndrome (SARS) virus. The similarities of 2019-nCoV and SARS virus suggested that the S and ORF6 proteins shared a low similarity, while the E protein shared the higher similarity. The 2019-nCoV sequence has similar potential phosphorylation sites and glycosylation sites on the surface protein and the ORF1ab polyprotein as the SARS virus; however, there are differences in potential modification sites between the Chinese strain and some American strains. At the same time, we proposed two possible recombination sites for 2019-nCoV. Based on the results of the skyline, we speculate that the activity of the gene population of 2019-nCoV may be before the end of 2019. As the scope of the 2019-nCoV infection further expands, it may produce different adaptive evolutions due to different environments. Finally, evolutionary genetic analysis can be a useful resource for studying the spread and virulence of 2019-nCoV, which are essential aspects of preventive and precise medicine.

Analysis of Three Mutations in Italian Strains of SARS-CoV-2: Implications for Pathogenesis.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus initially detected in Wuhan in December 2019, responsible for coronavirus disease 2019 (COVID-19), a respiratory syndrome currently affecting >220 countries around the world, with >80 million cases registered and >1.8 million deaths. OBJECTIVE: As several vaccines are still being developed and 2 have been approved, it is particularly important to perform evolutionary surveillance to identify mutations potentially affecting vaccine efficacy. METHODS: DynaMut server has been used to evaluate the impact of the mutation found on SARS-CoV-2 isolates available on GISAID. RESULTS: In this article, we analyze whole genomes sequenced from Italian patients, and we report the characterization of 3 mutations, one of which presents in the spike protein. CONCLUSION: The mutations analyzed in this article can be useful to evaluate the evolution of SARS-CoV-2.

Evolutionary analysis and lineage designation of SARS-CoV-2 genomes.

The pandemic due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has caused immense global disruption. With the rapid accumulation of SARS-CoV-2 genome sequences, however, thousands of genomic variants of SARS-CoV-2 are now publicly available. To improve the tracing of the viral genomes' evolution during the development of the pandemic, we analyzed single nucleotide variants (SNVs) in 121,618 high-quality SARS-CoV-2 genomes. We divided these viral genomes into two major lineages (L and S) based on variants at sites 8782 and 28144, and further divided the L lineage into two major sublineages (L1 and L2) using SNVs at sites 3037, 14408, and 23403. Subsequently, we categorized them into 130 sublineages (37 in S, 35 in L1, and 58 in L2) based on marker SNVs at 201 additional genomic sites. This lineage/sublineage designation system has a hierarchical structure and reflects the relatedness among the subclades of the major lineages. We also provide a companion website (www.covid19evolution.net) that allows users to visualize sublineage information and upload their own SARS-CoV-2 genomes for sublineage classification. Finally, we discussed the possible roles of compensatory mutations and natural selection during SARS-CoV-2's evolution. These efforts will improve our understanding of the temporal and spatial dynamics of SARS-CoV-2's genome evolution.

Isolating SARS-CoV-2 Strains From Countries in the Same Meridian: Genome Evolutionary Analysis.

BACKGROUND: COVID-19, caused by the novel SARS-CoV-2, is considered the most threatening respiratory infection in the world, with over 40 million people infected and over 0.934 million related deaths reported worldwide. It is speculated that epidemiological and clinical features of COVID-19 may differ across countries or continents. Genomic comparison of 48,635 SARS-CoV-2 genomes has shown that the average number of mutations per sample was 7.23, and most SARS-CoV-2 strains belong to one of 3 clades characterized by geographic and genomic specificity: Europe, Asia, and North America. OBJECTIVE: The aim of this study was to compare the genomes of SARS-CoV-2 strains isolated from Italy, Sweden, and Congo, that is, 3 different countries in the same meridian (longitude) but with different climate conditions, and from Brazil (as an outgroup country), to analyze similarities or differences in patterns of possible evolutionary pressure signatures in their genomes. METHODS: We obtained data from the Global Initiative on Sharing All Influenza Data repository by sampling all genomes available on that date. Using HyPhy, we achieved the recombination analysis by genetic algorithm recombination detection method, trimming, removal of the stop codons, and phylogenetic tree and mixed effects model of evolution analyses. We also performed secondary structure prediction analysis for both sequences (mutated and wild-type) and "disorder" and "transmembrane" analyses of the protein. We analyzed both protein structures with an ab initio approach to predict their ontologies and 3D structures. RESULTS: Evolutionary analysis revealed that codon 9628 is under episodic selective pressure for all SARS-CoV-2 strains isolated from the 4 countries, suggesting it is a key site for virus evolution. Codon 9628 encodes the P0DTD3 (Y14_SARS2) uncharacterized protein 14. Further investigation showed that the codon mutation was responsible for helical modification in the secondary structure. The codon was positioned in the more ordered region of the gene (41-59) and near to the area acting as the transmembrane (54-67), suggesting its involvement in the attachment phase of the virus. The predicted protein structures of both wild-type and mutated P0DTD3 confirmed the importance of the codon to define the protein structure. Moreover, ontological analysis of the protein emphasized that the mutation enhances the binding probability. CONCLUSIONS: Our results suggest that RNA secondary structure may be affected and, consequently, the protein product changes T (threonine) to G (glycine) in position 50 of the protein. This position is located close to the predicted transmembrane region. Mutation analysis revealed that the change from G (glycine) to D (aspartic acid) may confer a new function to the protein-binding activity, which in turn may be responsible for attaching the virus to human eukaryotic cells. These findings can help design in vitro experiments and possibly facilitate a vaccine design and successful antiviral strategies.

Territorywide Study of Early Coronavirus Disease Outbreak, Hong Kong, China.

Initial cases of coronavirus disease in Hong Kong were imported from mainland China. A dramatic increase in case numbers was seen in February 2020. Most case-patients had no recent travel history, suggesting the presence of transmission chains in the local community. We collected demographic, clinical, and epidemiologic data from 50 patients, who accounted for 53.8% of total reported case-patients as of February 28, 2020. We performed whole-genome sequencing to determine phylogenetic relationship and transmission dynamics of severe acute respiratory syndrome coronavirus 2 infections. By using phylogenetic analysis, we attributed the community outbreak to 2 lineages; 1 harbored a common mutation, Orf3a-G251V, and accounted for 88.0% of the cases in our study. The estimated time to the most recent common ancestor of local coronavirus disease outbreak was December 24, 2019, with an evolutionary rate of 3.04 × 10-3 substitutions/site/year. The reproduction number was 1.84, indicating ongoing community spread.

Ancestral origin, antigenic resemblance and epidemiological insights of novel coronavirus (SARS-CoV-2): Global burden and Bangladesh perspective.

SARS-CoV-2, a new coronavirus strain responsible for COVID-19, has emerged in Wuhan City, China, and continuing its global pandemic nature. The availability of the complete gene sequences of the virus helps to know about the origin and molecular characteristics of this virus. In the present study, we performed bioinformatic analysis of the available gene sequence data of SARS-CoV-2 for the understanding of evolution and molecular characteristics and immunogenic resemblance of the circulating viruses. Phylogenetic analysis was performed for four types of representative viral proteins (spike, membrane, envelope and nucleoprotein) of SARS-CoV-2, HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HKU1, MERS-CoV, HKU4, HKU5 and BufCoV-HKU26. The findings demonstrated that SARS-CoV-2 exhibited convergent evolutionary relation with previously reported SARS-CoV. It was also depicted that SARS-CoV-2 proteins were highly similar and identical to SARS-CoV proteins, though proteins from other coronaviruses showed a lower level of resemblance. The cross-checked conservancy analysis of SARS-CoV-2 antigenic epitopes showed significant conservancy with antigenic epitopes derived from SARS-CoV. Descriptive epidemiological analysis on several epidemiological indices was performed on available epidemiological outbreak information from several open databases on COVID-19 (SARS-CoV-2). Satellite-derived imaging data have been employed to understand the role of temperature in the environmental persistence of the virus. Findings of the descriptive analysis were used to describe the global impact of newly emerged SARS-CoV-2, and the risk of an epidemic in Bangladesh.

Phyloevolutionary analysis of SARS-CoV-2 in Nigeria.

Phyloepidemiological approaches have provided specific insight into understanding the emergence and evolution of infection. Knowledge on the outbreak and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Nigeria would assist in provision of preventive measures to reduce transmission among populations at risk. This study aimed to investigate the evolution of SARS-CoV-2 in Nigeria. A total of 39 complete genomes of SARS-CoV-2 were retrieved from the GISAID EpiFlu™ database on 29 March 2020 to investigate its evolution in Nigeria. Sequences were selected based on the travel history of the individual and the collection date. Other sequences were not selected because they were short, contained artefacts, were not from an original source or had insufficient information. Evolutionary history was inferred using the maximum likelihood method based on the general time reversible model. A phylogenetic tree was constructed to determine the common ancestor of each strain. The phylogenetic analysis showed that the strain in Nigeria clustered in a monophyletic clade with a Wuhan sublineage. Nucleotide alignment also showed a 100% similarity indicating a common origin of evolution. Comparative analysis showed 27 972 (93.6%) identical sites and 97.6% pairwise identity with the consensus. The study evidently showed the entire outbreak of SARS-CoV-2 infection in Nigeria stemmed from a single introduction sharing consensus similarity with the reference SARS-CoV-2 human genome from Wuhan. Preventive measures that can limit the spread of the infection among populations at risk should be implemented.

Structural and Evolutionary Analysis Indicate That the SARS-CoV-2 Mpro Is a Challenging Target for Small-Molecule Inhibitor Design.

The novel coronavirus whose outbreak took place in December 2019 continues to spread at a rapid rate worldwide. In the absence of an effective vaccine, inhibitor repurposing or de novo drug design may offer a longer-term strategy to combat this and future infections due to similar viruses. Here, we report on detailed classical and mixed-solvent molecular dynamics simulations of the main protease (Mpro) enriched by evolutionary and stability analysis of the protein. The results were compared with those for a highly similar severe acute respiratory syndrome (SARS) Mpro protein. In spite of a high level of sequence similarity, the active sites in both proteins showed major differences in both shape and size, indicating that repurposing SARS drugs for COVID-19 may be futile. Furthermore, analysis of the binding site's conformational changes during the simulation time indicated its flexibility and plasticity, which dashes hopes for rapid and reliable drug design. Conversely, structural stability of the protein with respect to flexible loop mutations indicated that the virus' mutability will pose a further challenge to the rational design of small-molecule inhibitors. However, few residues contribute significantly to the protein stability and thus can be considered as key anchoring residues for Mpro inhibitor design.