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1.
Nucleic Acids Res ; 50(5): 2509-2521, 2022 03 21.
Article in English | MEDLINE | ID: covidwho-1722548

ABSTRACT

Upon SARS-CoV-2 infection, viral intermediates specifically activate the IFN response through MDA5-mediated sensing and accordingly induce ADAR1 p150 expression, which might lead to viral A-to-I RNA editing. Here, we developed an RNA virus-specific editing identification pipeline, surveyed 7622 RNA-seq data from diverse types of samples infected with SARS-CoV-2, and constructed an atlas of A-to-I RNA editing sites in SARS-CoV-2. We found that A-to-I editing was dynamically regulated, varied between tissue and cell types, and was correlated with the intensity of innate immune response. On average, 91 editing events were deposited at viral dsRNA intermediates per sample. Moreover, editing hotspots were observed, including recoding sites in the spike gene that affect viral infectivity and antigenicity. Finally, we provided evidence that RNA editing accelerated SARS-CoV-2 evolution in humans during the epidemic. Our study highlights the ability of SARS-CoV-2 to hijack components of the host antiviral machinery to edit its genome and fuel its evolution, and also provides a framework and resource for studying viral RNA editing.


Subject(s)
COVID-19/immunology , Immunity, Innate/immunology , RNA Editing/immunology , SARS-CoV-2/immunology , Adenosine Deaminase/genetics , Adenosine Deaminase/immunology , Adenosine Deaminase/metabolism , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Base Sequence , Binding Sites/genetics , COVID-19/genetics , COVID-19/virology , Evolution, Molecular , Gene Expression/immunology , Humans , Immunity, Innate/genetics , Interferon-Induced Helicase, IFIH1/genetics , Interferon-Induced Helicase, IFIH1/immunology , Interferon-Induced Helicase, IFIH1/metabolism , Mutation , Protein Binding , RNA Editing/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/immunology , RNA-Binding Proteins/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Sequence Homology, Nucleic Acid , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
2.
Biochim Biophys Acta Mol Basis Dis ; 1868(2): 166291, 2022 02 01.
Article in English | MEDLINE | ID: covidwho-1525693

ABSTRACT

OBJECTIVES: To investigate in silico the presence of nucleotide sequence complementarity between the RNA genome of Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) and human non-coding (nc)RNA genes. METHODS: The FASTA sequence (NC_045512.2) of each of the 11 SARS-CoV-2 isolate Wuhan-Hu-1 genes was retrieved from NCBI.nlm.nih.gov/gene and the Ensembl.org library interrogated for any base-pair match with human ncRNA genes. SARS-CoV-2 gene-matched human ncRNAs were screened for functional activity using bioinformatic analysis. Finally, associations between identified ncRNAs and human diseases were searched in GWAS databases. RESULTS: A total of 252 matches were found between the nucleotide sequence of SARS-CoV-2 genes and human ncRNAs. With the exception of two small nuclear RNAs, all of them were long non-coding (lnc)RNAs expressed mainly in testis and central nervous system under physiological conditions. The percentage of alignment ranged from 91.30% to 100% with a mean nucleotide alignment length of 17.5 ± 2.4. Thirty-three (13.09%) of them contained predicted R-loop forming sequences, but none of these intersected the complementary sequences of SARS-CoV-2. However, in 31 cases matches fell on ncRNA regulatory sites, whose adjacent coding genes are mostly involved in cancer, immunological and neurological pathways. Similarly, several polymorphic variants of detected non-coding genes have been associated with neuropsychiatric and proliferative disorders. CONCLUSION: This pivotal in silico study shows that SARS-CoV-2 genes have Watson-Crick nucleotide complementarity to human ncRNA sequences, potentially disrupting ncRNA epigenetic control of target genes. It remains to be elucidated whether this could result in the development of human disease in the long term.


Subject(s)
COVID-19/genetics , COVID-19/virology , RNA, Untranslated/genetics , SARS-CoV-2/genetics , Base Sequence , Epigenesis, Genetic , Genes, Viral , Humans , Neoplasms/genetics , RNA, Long Noncoding/genetics , Sequence Alignment , Sequence Homology, Nucleic Acid
3.
Sci Rep ; 11(1): 19161, 2021 09 27.
Article in English | MEDLINE | ID: covidwho-1440480

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is associated with fatal pulmonary fibrosis. Small interfering RNAs (siRNAs) can be developed to induce RNA interference against SARS-CoV-2, and their susceptible target sites can be inferred by Argonaute crosslinking immunoprecipitation sequencing (AGO CLIP). Here, by reanalysing AGO CLIP data in RNA viruses, we delineated putative AGO binding in the conserved non-structural protein 12 (nsp12) region encoding RNA-dependent RNA polymerase (RdRP) in SARS-CoV-2. We utilised the inferred AGO binding to optimise the local RNA folding parameter to calculate target accessibility and predict all potent siRNA target sites in the SARS-CoV-2 genome, avoiding sequence variants. siRNAs loaded onto AGO also repressed seed (positions 2-8)-matched transcripts by acting as microRNAs (miRNAs). To utilise this, we further screened 13 potential siRNAs whose seed sequences were matched to known antifibrotic miRNAs and confirmed their miRNA-like activity. A miR-27-mimicking siRNA designed to target the nsp12 region (27/RdRP) was validated to silence a synthesised nsp12 RNA mimic in lung cell lines and function as an antifibrotic miR-27 in regulating target transcriptomes related to TGF-ß signalling. siRNA sequences with an antifibrotic miRNA-like activity that could synergistically treat COVID-19 are available online ( http://clip.korea.ac.kr/covid19 ).


Subject(s)
Argonaute Proteins/genetics , COVID-19/prevention & control , MicroRNAs/genetics , RNA, Small Interfering/genetics , SARS-CoV-2/genetics , A549 Cells , Argonaute Proteins/metabolism , Base Sequence , Binding Sites/genetics , COVID-19/virology , Cell Line , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Gene Expression Profiling/methods , HeLa Cells , Humans , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/metabolism , RNA Interference , RNA-Seq/methods , SARS-CoV-2/physiology , Sequence Homology, Nucleic Acid
4.
Sci Rep ; 11(1): 16145, 2021 08 09.
Article in English | MEDLINE | ID: covidwho-1349686

ABSTRACT

The genetic element s2m has been acquired through horizontal transfer by many distantly related viruses, including the SARS-related coronaviruses. Here we show that s2m is evolutionarily conserved in these viruses. Though several lineages of severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) devoid of the element can be found, these variants seem to have been short lived, indicating that they were less evolutionary fit than their s2m-containing counterparts. On a species-level, however, there do not appear to be any losses and this pattern strongly suggests that the s2m element is essential to virus replication in SARS-CoV-2 and related viruses. Further experiments are needed to elucidate the function of s2m.


Subject(s)
Coronaviridae/genetics , Interspersed Repetitive Sequences/genetics , RNA, Viral/genetics , SARS-CoV-2/genetics , Virus Replication/genetics , Animals , Base Sequence , COVID-19/virology , Coronaviridae/classification , Evolution, Molecular , Gene Transfer, Horizontal , Humans , Phylogeny , SARS-CoV-2/physiology , Sequence Homology, Nucleic Acid , Species Specificity
5.
J Virol Methods ; 295: 114215, 2021 09.
Article in English | MEDLINE | ID: covidwho-1275556

ABSTRACT

BACKGROUND: This study aimed to evaluate the impact of four different reverse transcription quantitative PCR (RT-qPCR) master mixes on the performance of SARS-CoV-2 diagnostic PCRs using three primer/probe assays targeting the N gene (A, B and C). The dynamic range and lowest detected quantity was determined using a SARS-CoV-2 partial N gene RNA transcript dilution series (100,000-1 copy/µl) and verified using 72 nose and throat swabs, 29 of which tested positive for SARS-CoV-2 RNA. RESULTS: Assay C consistently detected the lowest quantity of partial N gene RNA transcript with all mastermixes. The Takara One Step PrimeScript™ III RT-PCR Kit mastermix enabled all primer pairs to detect the entire dynamic range evaluated, with the Qiagen Quantifast and Thermofisher TaqPath 1-Step kits also performing well. Sequences from all three primer/probe sets tested in this study (assay A, B and C) have 100 % homology to ≥97 % of the of SARS-CoV-2 sequences available up to 31st December 2020 (n = 291,483 sequences). CONCLUSIONS: This work demonstrates that specific assays (in this case assay C) can perform well in terms of dynamic range and lowest detected quantity regardless of the mastermix used. However we also show that, by choosing the most appropriate mastermix, poorer performing primer pairs are also able to detect all of the template dilutions investigated. This work increases the potential options when choosing assays for SARS-CoV-2 diagnosis and provides solutions to enable them to work with optimal analytical sensitivity.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , Coronavirus Nucleocapsid Proteins/genetics , SARS-CoV-2/isolation & purification , COVID-19/diagnosis , COVID-19 Nucleic Acid Testing/instrumentation , DNA Primers/genetics , Humans , Nose/virology , Pharynx/virology , Phosphoproteins/genetics , RNA, Viral/genetics , Reagent Kits, Diagnostic , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity , Sequence Homology, Nucleic Acid
6.
Genes Genomics ; 43(9): 1079-1086, 2021 09.
Article in English | MEDLINE | ID: covidwho-1274972

ABSTRACT

BACKGROUND: Several reports on the discovery of SARS-CoV-2 mutations and variations in Indonesia COVID-19 cases led to genomic dysregulation with the first pandemic cases in Wuhan, China. MicroRNA (miRNA) plays an important role in this genetic regulation and contributes to the enhancement of viral RNA binding through the host mRNA. OBJECTIVE: This research is aimed to detect miRNA targets of SARS-CoV-2 and examines their role in Indonesia cases against Wuhan cases. METHODS: SARS-CoV-2 sequences were obtained from GISAID ( https://www.gisaid.org/ ), NCBI ( https://ncbi.nlm.nih.gov ), and National Genomics Data Center ( https://bigd.big.ac.cn/gwh/ ) databases. MiRDB ( https://github.com/gbnegrini/mirdb-custom-target-search ) was used to annotate and predict target human mature miRNAs. For statistical analysis, we utilized a series chi-square test to obtain significant miRNA. DIANA-miRPath v3.0 ( http://www.microrna.gr/miRPathv3 ) analyzed the Gene Ontology of mature miRNAs. RESULT: The statistical results detected five significant miRNAs. Two miRNAs: hsa-miR-4778-5p and hsa-miR-4531 were consistently found in the majority of Wuhan samples, while they were only found in less than half of the Indonesia samples. The other three miRNA, hsa-miR-6844, hsa-miR-627-5p, and hsa-miR-3674, were discovered in most samples in both groups but with a significant difference ratio. Among these five significant miRNA targets, hsa-miR-6844 is the only miRNA that has an association with the ORF1ab gene of SARS-CoV-2. CONCLUSION: The Gene Ontology analysis of five significant miRNA targets indicates a significant role in inflammation and the immune system. The specific detection of host miRNAs in this study shows that there are differences in the characteristics of SARS-CoV-2 between Indonesia and Wuhan.


Subject(s)
COVID-19/genetics , MicroRNAs/genetics , SARS-CoV-2/genetics , COVID-19/epidemiology , COVID-19/virology , China , Humans , Indonesia , SARS-CoV-2/pathogenicity , Sequence Homology, Nucleic Acid
7.
J Med Virol ; 93(7): 4382-4391, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1263102

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has spread around the globe very rapidly. Previously, the evolution pattern and similarity among the COVID-19 causative organism severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and causative organisms of other similar infections have been determined using a single type of genetic marker in different studies. Herein, the SARS-CoV-2 and related ß coronaviruses Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV,  bat coronavirus (BAT-CoV) were comprehensively analyzed using a custom-built pipeline that employed phylogenetic approaches based on multiple types of genetic markers including the whole genome sequences, mutations in nucleotide sequences, mutations in protein sequences, and microsatellites. The whole-genome sequence-based phylogeny revealed that the strains of SARS-CoV-2 are more similar to the BAT-CoV strains. The mutational analysis showed that on average MERS-CoV and BAT-CoV genomes differed at 134.21 and 136.72 sites, respectively, whereas the SARS-CoV genome differed at 26.64 sites from the reference genome of SARS-CoV-2. Furthermore, the microsatellite analysis highlighted a relatively higher number of average microsatellites for MERS-CoV and SARS-CoV-2 (106.8 and 107, respectively), and a lower number for SARS-CoV and BAT-CoV (95.8 and 98.5, respectively). Collectively, the analysis of multiple genetic markers of selected ß viral genomes revealed that the newly born SARS-COV-2 is closely related to BAT-CoV, whereas, MERS-CoV is more distinct from the SARS-CoV-2 than BAT-CoV and SARS-CoV.


Subject(s)
Alphacoronavirus/genetics , Genome, Viral/genetics , Microsatellite Repeats/genetics , Middle East Respiratory Syndrome Coronavirus/genetics , SARS Virus/genetics , SARS-CoV-2/genetics , Animals , Base Sequence/genetics , Chiroptera/virology , DNA Mutational Analysis , Genetic Markers/genetics , Genetic Variation/genetics , Humans , Phylogeny , Sequence Alignment , Sequence Homology, Nucleic Acid , Whole Genome Sequencing
8.
J Med Virol ; 93(7): 4382-4391, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1156882

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has spread around the globe very rapidly. Previously, the evolution pattern and similarity among the COVID-19 causative organism severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and causative organisms of other similar infections have been determined using a single type of genetic marker in different studies. Herein, the SARS-CoV-2 and related ß coronaviruses Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV,  bat coronavirus (BAT-CoV) were comprehensively analyzed using a custom-built pipeline that employed phylogenetic approaches based on multiple types of genetic markers including the whole genome sequences, mutations in nucleotide sequences, mutations in protein sequences, and microsatellites. The whole-genome sequence-based phylogeny revealed that the strains of SARS-CoV-2 are more similar to the BAT-CoV strains. The mutational analysis showed that on average MERS-CoV and BAT-CoV genomes differed at 134.21 and 136.72 sites, respectively, whereas the SARS-CoV genome differed at 26.64 sites from the reference genome of SARS-CoV-2. Furthermore, the microsatellite analysis highlighted a relatively higher number of average microsatellites for MERS-CoV and SARS-CoV-2 (106.8 and 107, respectively), and a lower number for SARS-CoV and BAT-CoV (95.8 and 98.5, respectively). Collectively, the analysis of multiple genetic markers of selected ß viral genomes revealed that the newly born SARS-COV-2 is closely related to BAT-CoV, whereas, MERS-CoV is more distinct from the SARS-CoV-2 than BAT-CoV and SARS-CoV.


Subject(s)
Alphacoronavirus/genetics , Genome, Viral/genetics , Microsatellite Repeats/genetics , Middle East Respiratory Syndrome Coronavirus/genetics , SARS Virus/genetics , SARS-CoV-2/genetics , Animals , Base Sequence/genetics , Chiroptera/virology , DNA Mutational Analysis , Genetic Markers/genetics , Genetic Variation/genetics , Humans , Phylogeny , Sequence Alignment , Sequence Homology, Nucleic Acid , Whole Genome Sequencing
9.
Viruses ; 12(12)2020 12 21.
Article in English | MEDLINE | ID: covidwho-1000348

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. The 3' untranslated region (UTR) of this ß-CoV contains essential cis-acting RNA elements for the viral genome transcription and replication. These elements include an equilibrium between an extended bulged stem-loop (BSL) and a pseudoknot. The existence of such an equilibrium is supported by reverse genetic studies and phylogenetic covariation analysis and is further proposed as a molecular switch essential for the control of the viral RNA polymerase binding. Here, we report the SARS-CoV-2 3' UTR structures in cells that transcribe the viral UTRs harbored in a minigene plasmid and isolated infectious virions using a chemical probing technique, namely dimethyl sulfate (DMS)-mutational profiling with sequencing (MaPseq). Interestingly, the putative pseudoknotted conformation was not observed, indicating that its abundance in our systems is low in the absence of the viral nonstructural proteins (nsps). Similarly, our results also suggest that another functional cis-acting element, the three-helix junction, cannot stably form. The overall architectures of the viral 3' UTRs in the infectious virions and the minigene-transfected cells are almost identical.


Subject(s)
3' Untranslated Regions/genetics , COVID-19/virology , Nucleic Acid Conformation , Pandemics , RNA, Viral/genetics , SARS-CoV-2/genetics , Animals , Base Sequence , Cell Line , Conserved Sequence , Cricetinae , High-Throughput Nucleotide Sequencing , Humans , Mesocricetus , Models, Molecular , Plasmids , Point Mutation , Reverse Genetics/methods , SARS-CoV-2/physiology , Sequence Alignment , Sequence Homology, Nucleic Acid , Sulfuric Acid Esters , Transcription, Genetic , Virion/genetics , Virion/physiology
10.
Genome Res ; 30(10): 1434-1448, 2020 10.
Article in English | MEDLINE | ID: covidwho-963139

ABSTRACT

The human pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the major pandemic of the twenty-first century. We analyzed more than 4700 SARS-CoV-2 genomes and associated metadata retrieved from public repositories. SARS-CoV-2 sequences have a high sequence identity (>99.9%), which drops to >96% when compared to bat coronavirus genome. We built a mutation-annotated reference SARS-CoV-2 phylogeny with two main macro-haplogroups, A and B, both of Asian origin, and more than 160 sub-branches representing virus strains of variable geographical origins worldwide, revealing a rather uniform mutation occurrence along branches that could have implications for diagnostics and the design of future vaccines. Identification of the root of SARS-CoV-2 genomes is not without problems, owing to conflicting interpretations derived from either using the bat coronavirus genomes as an outgroup or relying on the sampling chronology of the SARS-CoV-2 genomes and TMRCA estimates; however, the overall scenario favors haplogroup A as the ancestral node. Phylogenetic analysis indicates a TMRCA for SARS-CoV-2 genomes dating to November 12, 2019, thus matching epidemiological records. Sub-haplogroup A2 most likely originated in Europe from an Asian ancestor and gave rise to subclade A2a, which represents the major non-Asian outbreak, especially in Africa and Europe. Multiple founder effect episodes, most likely associated with super-spreader hosts, might explain COVID-19 pandemic to a large extent.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/epidemiology , Genome, Viral/genetics , Pneumonia, Viral/epidemiology , Animals , Asia/epidemiology , Base Sequence/genetics , COVID-19 , Chiroptera/virology , Chromosome Mapping , Europe/epidemiology , Evolution, Molecular , Genetic Variation/genetics , Humans , Pandemics , Phylogeny , Phylogeography , SARS-CoV-2 , Sequence Homology, Nucleic Acid
11.
Euro Surveill ; 25(42)2020 10.
Article in English | MEDLINE | ID: covidwho-886129

ABSTRACT

An outbreak of 59 cases of coronavirus disease (COVID-19) originated with 13 cases linked by a 7 h, 17% occupancy flight into Ireland, summer 2020. The flight-associated attack rate was 9.8-17.8%. Spread to 46 non-flight cases occurred country-wide. Asymptomatic/pre-symptomatic transmission in-flight from a point source is implicated by 99% homology across the virus genome in five cases travelling from three different continents. Restriction of movement on arrival and robust contact tracing can limit propagation post-flight.


Subject(s)
Air Travel , Betacoronavirus/isolation & purification , Coronavirus Infections/epidemiology , Pandemics , Pneumonia, Viral/epidemiology , Travel-Related Illness , Asymptomatic Diseases , Betacoronavirus/genetics , COVID-19 , Contact Tracing , Coronavirus Infections/prevention & control , Coronavirus Infections/transmission , Coronavirus Infections/virology , Disease Outbreaks , Environmental Exposure , Family Characteristics , Forms and Records Control , Genome, Viral , Hospitalization , Humans , Infection Control/methods , Ireland/epidemiology , Nasopharynx/virology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , RNA, Viral/genetics , SARS-CoV-2 , Seasons , Sequence Homology, Nucleic Acid , Time Factors , Whole Genome Sequencing
12.
Elife ; 92020 10 01.
Article in English | MEDLINE | ID: covidwho-809713

ABSTRACT

Understanding the emergence of novel viruses requires an accurate and comprehensive annotation of their genomes. Overlapping genes (OLGs) are common in viruses and have been associated with pandemics but are still widely overlooked. We identify and characterize ORF3d, a novel OLG in SARS-CoV-2 that is also present in Guangxi pangolin-CoVs but not other closely related pangolin-CoVs or bat-CoVs. We then document evidence of ORF3d translation, characterize its protein sequence, and conduct an evolutionary analysis at three levels: between taxa (21 members of Severe acute respiratory syndrome-related coronavirus), between human hosts (3978 SARS-CoV-2 consensus sequences), and within human hosts (401 deeply sequenced SARS-CoV-2 samples). ORF3d has been independently identified and shown to elicit a strong antibody response in COVID-19 patients. However, it has been misclassified as the unrelated gene ORF3b, leading to confusion. Our results liken ORF3d to other accessory genes in emerging viruses and highlight the importance of OLGs.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Evolution, Molecular , Genes, Overlapping , Genes, Viral , Host Specificity/genetics , Open Reading Frames/genetics , Pandemics , Pneumonia, Viral/virology , Viral Proteins/genetics , Amino Acid Sequence , Animals , Antibodies, Viral/immunology , Antibody Specificity , Antigens, Viral/biosynthesis , Antigens, Viral/genetics , Antigens, Viral/immunology , Betacoronavirus/pathogenicity , Betacoronavirus/physiology , COVID-19 , China/epidemiology , Chiroptera/virology , Coronavirus/genetics , Coronavirus Infections/epidemiology , Epitopes/genetics , Epitopes/immunology , Europe/epidemiology , Eutheria/virology , Gene Expression Regulation, Viral , Genetic Variation , Haplotypes/genetics , Humans , Models, Molecular , Mutation , Phylogeny , Pneumonia, Viral/epidemiology , Protein Biosynthesis , Protein Conformation , RNA, Viral/genetics , SARS-CoV-2 , Sequence Alignment , Sequence Homology, Nucleic Acid , Viral Proteins/immunology
13.
In Vivo ; 34(3 Suppl): 1633-1636, 2020 Jun.
Article in English | MEDLINE | ID: covidwho-534631

ABSTRACT

In a previous study, we identified a 117 base severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequence in the human genome with 94.6% identity. The sequence was in chromosome 1p within an intronic region of the netrin G1 (NTNG1) gene. The sequence matched a sequence in the SARS-CoV-2 Orf1b gene in non-structural protein 14 (NSP14), which is an exonuclease and NSP15, an endoribonuclease. In the current study we compared the human genome with other viral genomes to determine some of the characteristics of human sequences found in the latter. Most of the viruses had human sequences, but they were short. Hepatitis A and St Louis encephalitis had human sequences that were longer than the 117 base SARS-Cov-2 sequence, but they were in non-coding regions of the human genome. The SARS-Cov-2 sequence was the only long sequence found in a human gene (NTNG1). The related coronaviruses SARS-Cov had a 41 BP human sequence on chromosome 3 that was not part of a human gene, and MERS had no human sequence. The 117 base SARS-CoV-2 human sequence is relatively close to the viral spike sequence, separated only by NSP16, a 904 base sequence. The mechanism for SARS-CoV-2 infection is the binding of the virus spike protein to the membrane-bound form of angiotensin-converting enzyme 2 (ACE2) and internalization of the complex by the host cell. We have no explanation for the NSP14 and NSP15 SARS-Cov-2 sequences we observed here or how they might relate to infectiousness. Further studies are warranted.


Subject(s)
Betacoronavirus/genetics , Exoribonucleases/genetics , Genome, Viral , SARS Virus/genetics , Viral Nonstructural Proteins/genetics , Chromosomes, Human, Pair 1/genetics , Chromosomes, Human, Pair 3/genetics , DNA Viruses/genetics , Endoribonucleases , GPI-Linked Proteins/genetics , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , Netrins/genetics , SARS-CoV-2 , Sequence Alignment , Sequence Homology, Nucleic Acid , Species Specificity , Viral Proteins/genetics
14.
In Vivo ; 34(3 Suppl): 1629-1632, 2020 Jun.
Article in English | MEDLINE | ID: covidwho-534630

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus. It is contagious in humans and is the cause of the coronavirus disease 2019 (COVID-19) pandemic. In the current analysis, we searched for SARS-CoV-2 sequences within the human genome. To compare the SARS-CoV-2 genome to the human genome, we used the blast-like alignment tool (BLAT) of the University of California, Santa Cruz Genome Browser. BLAT can align a user sequence of 25 bases or more to the genome. BLAT search results revealed a 117-base pair SARS-CoV-2 sequence in the human genome with 94.6% identity. The sequence was in chromosome 1p within an intronic region of the netrin G1 (NTNG1) gene. The sequence matched a sequence in the SARS-CoV-2 orf1b (open reading frames) gene. The SARS-CoV-2 human sequence lies within non-structural proteins 14 and 15 (NSP14 and NSP15), and is quite close to the viral spike sequence, separated only by NSP16, a 904-base pair sequence. The mechanism for SARS-CoV-2 infection is the binding of the virus spike protein to the membrane-bound form of angiotensin-converting enzyme 2 and internalization of the complex by the host cell. It is probably no accident that a sequence from the SARS-CoV-2 orf1b gene is found in the human NTNG1 gene, implicated in schizophrenia, and that haloperidol, used to treat schizophrenia, may also be a treatment for COVID-19. We suggest, therefore, that it is important to investigate other haloperidol analogs. Among them are benperidol, bromperidol, bromperidol decanoate, droperidol, seperidol hydrochloride, and trifluperidol. These analogs might be valuable in the treatment of COVID-19 and other coronavirus infections.


Subject(s)
Betacoronavirus/genetics , Chromosomes, Human, Pair 1/genetics , Exoribonucleases/genetics , Genes, Viral , Netrin-1/genetics , Viral Nonstructural Proteins/genetics , Viral Proteins/genetics , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Base Sequence , COVID-19 , Coronavirus Infections/drug therapy , DNA, Complementary/genetics , Endoribonucleases/genetics , Haloperidol/analogs & derivatives , Haloperidol/pharmacology , Haloperidol/therapeutic use , Humans , Introns/genetics , Pan troglodytes/genetics , Pandemics , Pneumonia, Viral/drug therapy , Polyproteins , RNA, Viral/genetics , SARS-CoV-2 , Schizophrenia/drug therapy , Schizophrenia/genetics , Sequence Alignment , Sequence Homology, Nucleic Acid , Species Specificity
15.
Int J Mol Sci ; 21(11)2020 May 29.
Article in English | MEDLINE | ID: covidwho-436912

ABSTRACT

Advances in sequencing technology have made large amounts of biological data available. Evolutionary analysis of data such as DNA sequences is highly important in biological studies. As alignment methods are ineffective for analyzing large-scale data due to their inherently high costs, alignment-free methods have recently attracted attention in the field of bioinformatics. In this paper, we introduce a new positional correlation natural vector (PCNV) method that involves converting a DNA sequence into an 18-dimensional numerical feature vector. Using frequency and position correlation to represent the nucleotide distribution, it is possible to obtain a PCNV for a DNA sequence. This new numerical vector design uses six suitable features to characterize the correlation among nucleotide positions in sequences. PCNV is also very easy to compute and can be used for rapid genome comparison. To test our novel method, we performed phylogenetic analysis with several viral and bacterial genome datasets with PCNV. For comparison, an alignment-based method, Bayesian inference, and two alignment-free methods, feature frequency profile and natural vector, were performed using the same datasets. We found that the PCNV technique is fast and accurate when used for phylogenetic analysis and classification of viruses and bacteria.


Subject(s)
Phylogeny , Sequence Analysis, DNA/methods , Sequence Homology, Nucleic Acid , Algorithms , Genome, Bacterial , Genome, Viral , Sequence Alignment
16.
PLoS Pathog ; 16(5): e1008421, 2020 05.
Article in English | MEDLINE | ID: covidwho-260623

ABSTRACT

The outbreak of a novel corona Virus Disease 2019 (COVID-19) in the city of Wuhan, China has resulted in more than 1.7 million laboratory confirmed cases all over the world. Recent studies showed that SARS-CoV-2 was likely originated from bats, but its intermediate hosts are still largely unknown. In this study, we assembled the complete genome of a coronavirus identified in 3 sick Malayan pangolins. The molecular and phylogenetic analyses showed that this pangolin coronavirus (pangolin-CoV-2020) is genetically related to the SARS-CoV-2 as well as a group of bat coronaviruses but do not support the SARS-CoV-2 emerged directly from the pangolin-CoV-2020. Our study suggests that pangolins are natural hosts of Betacoronaviruses. Large surveillance of coronaviruses in pangolins could improve our understanding of the spectrum of coronaviruses in pangolins. In addition to conservation of wildlife, minimizing the exposures of humans to wildlife will be important to reduce the spillover risks of coronaviruses from wild animals to humans.


Subject(s)
Betacoronavirus/classification , Betacoronavirus/genetics , Coronavirus Infections/virology , Disease Reservoirs/virology , Eutheria/virology , Pneumonia, Viral/virology , Animals , COVID-19 , Coronaviridae/classification , Coronaviridae/genetics , Host Specificity , Humans , Pandemics , Phylogeny , SARS-CoV-2 , Sequence Homology, Nucleic Acid , Zoonoses/prevention & control , Zoonoses/virology
17.
Nature ; 583(7815): 286-289, 2020 07.
Article in English | MEDLINE | ID: covidwho-210764

ABSTRACT

The current outbreak of coronavirus disease-2019 (COVID-19) poses unprecedented challenges to global health1. The new coronavirus responsible for this outbreak-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-shares high sequence identity to SARS-CoV and a bat coronavirus, RaTG132. Although bats may be the reservoir host for a variety of coronaviruses3,4, it remains unknown whether SARS-CoV-2 has additional host species. Here we show that a coronavirus, which we name pangolin-CoV, isolated from a Malayan pangolin has 100%, 98.6%, 97.8% and 90.7% amino acid identity with SARS-CoV-2 in the E, M, N and S proteins, respectively. In particular, the receptor-binding domain of the S protein of pangolin-CoV is almost identical to that of SARS-CoV-2, with one difference in a noncritical amino acid. Our comparative genomic analysis suggests that SARS-CoV-2 may have originated in the recombination of a virus similar to pangolin-CoV with one similar to RaTG13. Pangolin-CoV was detected in 17 out of the 25 Malayan pangolins that we analysed. Infected pangolins showed clinical signs and histological changes, and circulating antibodies against pangolin-CoV reacted with the S protein of SARS-CoV-2. The isolation of a coronavirus from pangolins that is closely related to SARS-CoV-2 suggests that these animals have the potential to act as an intermediate host of SARS-CoV-2. This newly identified coronavirus from pangolins-the most-trafficked mammal in the illegal wildlife trade-could represent a future threat to public health if wildlife trade is not effectively controlled.


Subject(s)
Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Eutheria/virology , Evolution, Molecular , Genome, Viral/genetics , Sequence Homology, Nucleic Acid , Animals , Betacoronavirus/classification , COVID-19 , China , Chiroptera/virology , Chlorocebus aethiops , Coronavirus Envelope Proteins , Coronavirus Infections/epidemiology , Coronavirus Infections/pathology , Coronavirus Infections/transmission , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Coronavirus M Proteins , Coronavirus Nucleocapsid Proteins , Disease Reservoirs/virology , Genomics , Host Specificity , Humans , Lung/pathology , Lung/virology , Malaysia , Nucleocapsid Proteins/genetics , Pandemics , Phosphoproteins , Phylogeny , Pneumonia, Viral/epidemiology , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , Polymerase Chain Reaction , Recombination, Genetic , SARS-CoV-2 , Sequence Alignment , Sequence Analysis, RNA , Spike Glycoprotein, Coronavirus/genetics , Vero Cells , Viral Envelope Proteins/genetics , Viral Matrix Proteins/genetics , Zoonoses/transmission , Zoonoses/virology
18.
Nature ; 583(7815): 282-285, 2020 07.
Article in English | MEDLINE | ID: covidwho-17844

ABSTRACT

The ongoing outbreak of viral pneumonia in China and across the world is associated with a new coronavirus, SARS-CoV-21. This outbreak has been tentatively associated with a seafood market in Wuhan, China, where the sale of wild animals may be the source of zoonotic infection2. Although bats are probable reservoir hosts for SARS-CoV-2, the identity of any intermediate host that may have facilitated transfer to humans is unknown. Here we report the identification of SARS-CoV-2-related coronaviruses in Malayan pangolins (Manis javanica) seized in anti-smuggling operations in southern China. Metagenomic sequencing identified pangolin-associated coronaviruses that belong to two sub-lineages of SARS-CoV-2-related coronaviruses, including one that exhibits strong similarity in the receptor-binding domain to SARS-CoV-2. The discovery of multiple lineages of pangolin coronavirus and their similarity to SARS-CoV-2 suggests that pangolins should be considered as possible hosts in the emergence of new coronaviruses and should be removed from wet markets to prevent zoonotic transmission.


Subject(s)
Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Eutheria/virology , Evolution, Molecular , Genome, Viral/genetics , Sequence Homology, Nucleic Acid , Amino Acid Sequence , Animals , Betacoronavirus/chemistry , Betacoronavirus/classification , COVID-19 , China/epidemiology , Chiroptera/virology , Coronavirus Infections/epidemiology , Coronavirus Infections/transmission , Coronavirus Infections/virology , Disease Reservoirs/virology , Genomics , Humans , Malaysia , Pandemics , Phylogeny , Pneumonia, Viral/epidemiology , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , Recombination, Genetic , SARS-CoV-2 , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Zoonoses/virology
19.
J Med Virol ; 92(6): 667-674, 2020 06.
Article in English | MEDLINE | ID: covidwho-8510

ABSTRACT

Starting around December 2019, an epidemic of pneumonia, which was named COVID-19 by the World Health Organization, broke out in Wuhan, China, and is spreading throughout the world. A new coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses was soon found to be the cause. At present, the sensitivity of clinical nucleic acid detection is limited, and it is still unclear whether it is related to genetic variation. In this study, we retrieved 95 full-length genomic sequences of SARAS-CoV-2 strains from the National Center for Biotechnology Information and GISAID databases, established the reference sequence by conducting multiple sequence alignment and phylogenetic analyses, and analyzed sequence variations along the SARS-CoV-2 genome. The homology among all viral strains was generally high, among them, 99.99% (99.91%-100%) at the nucleotide level and 99.99% (99.79%-100%) at the amino acid level. Although overall variation in open-reading frame (ORF) regions is low, 13 variation sites in 1a, 1b, S, 3a, M, 8, and N regions were identified, among which positions nt28144 in ORF 8 and nt8782 in ORF 1a showed mutation rate of 30.53% (29/95) and 29.47% (28/95), respectively. These findings suggested that there may be selective mutations in SARS-COV-2, and it is necessary to avoid certain regions when designing primers and probes. Establishment of the reference sequence for SARS-CoV-2 could benefit not only biological study of this virus but also diagnosis, clinical monitoring and intervention of SARS-CoV-2 infection in the future.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Coronavirus Infections/epidemiology , Genome, Viral , Mutation Rate , Pandemics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/epidemiology , Base Sequence , Betacoronavirus/classification , Betacoronavirus/isolation & purification , Betacoronavirus/pathogenicity , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/methods , Coronavirus Infections/transmission , Coronavirus Infections/virology , Databases, Genetic , Humans , Open Reading Frames , Phylogeny , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , RNA, Viral/genetics , Reference Standards , SARS-CoV-2 , Sequence Alignment , Sequence Homology, Nucleic Acid
20.
J Med Virol ; 92(6): 660-666, 2020 06.
Article in English | MEDLINE | ID: covidwho-7544

ABSTRACT

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging disease with fatal outcomes. In this study, a fundamental knowledge gap question is to be resolved by evaluating the differences in biological and pathogenic aspects of SARS-CoV-2 and the changes in SARS-CoV-2 in comparison with the two prior major COV epidemics, SARS and Middle East respiratory syndrome (MERS) coronaviruses. METHODS: The genome composition, nucleotide analysis, codon usage indices, relative synonymous codons usage, and effective number of codons (ENc) were analyzed in the four structural genes; Spike (S), Envelope (E), membrane (M), and Nucleocapsid (N) genes, and two of the most important nonstructural genes comprising RNA-dependent RNA polymerase and main protease (Mpro) of SARS-CoV-2, Beta-CoV from pangolins, bat SARS, MERS, and SARS CoVs. RESULTS: SARS-CoV-2 prefers pyrimidine rich codons to purines. Most high-frequency codons were ending with A or T, while the low frequency and rare codons were ending with G or C. SARS-CoV-2 structural proteins showed 5 to 20 lower ENc values, compared with SARS, bat SARS, and MERS CoVs. This implies higher codon bias and higher gene expression efficiency of SARS-CoV-2 structural proteins. SARS-CoV-2 encoded the highest number of over-biased and negatively biased codons. Pangolin Beta-CoV showed little differences with SARS-CoV-2 ENc values, compared with SARS, bat SARS, and MERS CoV. CONCLUSION: Extreme bias and lower ENc values of SARS-CoV-2, especially in Spike, Envelope, and Mpro genes, are suggestive for higher gene expression efficiency, compared with SARS, bat SARS, and MERS CoVs.


Subject(s)
Betacoronavirus/genetics , Cysteine Endopeptidases/genetics , Middle East Respiratory Syndrome Coronavirus/genetics , Nucleocapsid Proteins/genetics , RNA-Dependent RNA Polymerase/genetics , SARS Virus/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Envelope Proteins/genetics , Viral Nonstructural Proteins/genetics , Animals , Base Sequence , Betacoronavirus/classification , Betacoronavirus/pathogenicity , COVID-19 , Chiroptera/microbiology , Codon Usage , Computational Biology , Coronavirus 3C Proteases , Coronavirus Envelope Proteins , Coronavirus Infections/epidemiology , Coronavirus Infections/transmission , Coronavirus Infections/virology , Coronavirus Nucleocapsid Proteins , Cysteine Endopeptidases/metabolism , Eutheria/microbiology , Gene Expression , Humans , Middle East Respiratory Syndrome Coronavirus/classification , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Nucleocapsid Proteins/metabolism , Pandemics , Phosphoproteins , Pneumonia, Viral/epidemiology , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , RNA-Dependent RNA Polymerase/metabolism , SARS Virus/classification , SARS Virus/pathogenicity , SARS-CoV-2 , Sequence Homology, Nucleic Acid , Severe Acute Respiratory Syndrome/epidemiology , Severe Acute Respiratory Syndrome/transmission , Severe Acute Respiratory Syndrome/virology , Spike Glycoprotein, Coronavirus/metabolism , Viral Envelope Proteins/metabolism , Viral Nonstructural Proteins/metabolism
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