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1.
Indian J Med Res ; 151(2 & 3): 200-209, 2020.
Article in English | MEDLINE | ID: covidwho-1726321

ABSTRACT

Background & objectives: Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally affected 195 countries. In India, suspected cases were screened for SARS-CoV-2 as per the advisory of the Ministry of Health and Family Welfare. The objective of this study was to characterize SARS-CoV-2 sequences from three identified positive cases as on February 29, 2020. Methods: Throat swab/nasal swab specimens for a total of 881 suspected cases were screened by E gene and confirmed by RdRp (1), RdRp (2) and N gene real-time reverse transcription-polymerase chain reactions and next-generation sequencing. Phylogenetic analysis, molecular characterization and prediction of B- and T-cell epitopes for Indian SARS-CoV-2 sequences were undertaken. Results: Three cases with a travel history from Wuhan, China, were confirmed positive for SARS-CoV-2. Almost complete (29,851 nucleotides) genomes of case 1, case 3 and a fragmented genome for case 2 were obtained. The sequences of Indian SARS-CoV-2 though not identical showed high (~99.98%) identity with Wuhan seafood market pneumonia virus (accession number: NC 045512). Phylogenetic analysis showed that the Indian sequences belonged to different clusters. Predicted linear B-cell epitopes were found to be concentrated in the S1 domain of spike protein, and a conformational epitope was identified in the receptor-binding domain. The predicted T-cell epitopes showed broad human leucocyte antigen allele coverage of A and B supertypes predominant in the Indian population. Interpretation & conclusions: The two SARS-CoV-2 sequences obtained from India represent two different introductions into the country. The genetic heterogeneity is as noted globally. The identified B- and T-cell epitopes may be considered suitable for future experiments towards the design of vaccines and diagnostics. Continuous monitoring and analysis of the sequences of new cases from India and the other affected countries would be vital to understand the genetic evolution and rates of substitution of the SARS-CoV-2.


Subject(s)
Betacoronavirus/genetics , Genome, Viral , COVID-19 , Coronavirus Infections , Epitopes, B-Lymphocyte/genetics , Epitopes, T-Lymphocyte/genetics , Humans , India , Models, Molecular , Pandemics , Phylogeny , Pneumonia, Viral , Protein Structure, Tertiary , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics
2.
Clin Infect Dis ; 74(2): 237-245, 2022 01 29.
Article in English | MEDLINE | ID: covidwho-1662115

ABSTRACT

BACKGROUND: Both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection and persistent infection have been reported, but sequence characteristics in these scenarios have not been described. We assessed published cases of SARS-CoV-2 reinfection and persistence, characterizing the hallmarks of reinfecting sequences and the rate of viral evolution in persistent infection. METHODS: A systematic review of PubMed was conducted to identify cases of SARS-CoV-2 reinfection and persistence with available sequences. Nucleotide and amino acid changes in the reinfecting sequence were compared with both the initial and contemporaneous community variants. Time-measured phylogenetic reconstruction was performed to compare intrahost viral evolution in persistent SARS-CoV-2 to community-driven evolution. RESULTS: Twenty reinfection and 9 persistent infection cases were identified. Reports of reinfection cases spanned a broad distribution of ages, baseline health status, reinfection severity, and occurred as early as 1.5 months or >8 months after the initial infection. The reinfecting viral sequences had a median of 17.5 nucleotide changes with enrichment in the ORF8 and N genes. The number of changes did not differ by the severity of reinfection and reinfecting variants were similar to the contemporaneous sequences circulating in the community. Patients with persistent coronavirus disease 2019 (COVID-19) demonstrated more rapid accumulation of sequence changes than seen with community-driven evolution with continued evolution during convalescent plasma or monoclonal antibody treatment. CONCLUSIONS: Reinfecting SARS-CoV-2 viral genomes largely mirror contemporaneous circulating sequences in that geographic region, while persistent COVID-19 has been largely described in immunosuppressed individuals and is associated with accelerated viral evolution.


Subject(s)
COVID-19 , SARS-CoV-2 , COVID-19/therapy , Humans , Immunization, Passive , Infant , Phylogeny , Reinfection
3.
Jpn J Infect Dis ; 74(5): 465-472, 2021 Sep 22.
Article in English | MEDLINE | ID: covidwho-1436361

ABSTRACT

Soon after the 2019 outbreak of coronavirus disease 2019 in Wuhan, China, a protocol for real-time RT-PCR assay detection of severe acute respiratory syndrome coronavirus (SARS-CoV-2) was established by the National Institute of Infectious Diseases (NIID) in Japan. The protocol used Charité's nucleocapsid (Sarbeco-N) and NIID nucleocapsid (NIID-N2) assays. During the following months, SARS-CoV-2 spread and caused a global pandemic, and various SARS-CoV-2 sequences were registered in public databases, such as the Global Initiative on Sharing All Influenza Data (GISAID). In this study, we evaluated the S2 assay (NIID-S2) that was newly developed to replace the Sarbeco-N assay and the performance of the NIID-N2 and NIID-S2 assays, referring to mismatches in the primer/probe targeted region. We found that the analytical sensitivity and specificity of the NIID-S2 set were comparable to those of the NIID-N2 assay, and the detection rate for clinical specimens was identical to that of the NIID-N2 assay. Furthermore, among the available sequences (approximately 192,000), the NIID-N2 and NIID-S2 sets had 2.6% and 1.2% mismatched sequences, respectively, although most of these mismatches did not affect the amplification efficiency, except the 3' end of the NIID-N2 forward primer. These findings indicate that the previously developed NIID-N2 assay is suitable for the detection of SARS-CoV-2 with support from the newly developed NIID-S2 set.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , Coronavirus Nucleocapsid Proteins/genetics , DNA Primers/genetics , Humans , Japan , Phosphoproteins/genetics , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics
4.
Genomics ; 113(4): 1628-1638, 2021 07.
Article in English | MEDLINE | ID: covidwho-1386752

ABSTRACT

Sequencing the SARS-CoV-2 genome from clinical samples can be challenging, especially in specimens with low viral titer. Here we report Accurate SARS-CoV-2 genome Reconstruction (ACoRE), an amplicon-based viral genome sequencing workflow for the complete and accurate reconstruction of SARS-CoV-2 sequences from clinical samples, including suboptimal ones that would usually be excluded even if unique and irreplaceable. The protocol was optimized to improve flexibility and the combination of technical replicates was established as the central strategy to achieve accurate analysis of low-titer/suboptimal samples. We demonstrated the utility of the approach by achieving complete genome reconstruction and the identification of false-positive variants in >170 clinical samples, thus avoiding the generation of inaccurate and/or incomplete sequences. Most importantly, ACoRE was crucial to identify the correct viral strain responsible of a relapse case, that would be otherwise mis-classified as a re-infection due to missing or incorrect variant identification by a standard workflow.


Subject(s)
COVID-19/genetics , Genome, Viral/genetics , Reinfection/genetics , SARS-CoV-2/genetics , COVID-19/pathology , COVID-19/virology , Genetic Variation/genetics , Humans , Reinfection/pathology , Reinfection/virology , SARS-CoV-2/pathogenicity , Whole Genome Sequencing
5.
Bioinformatics ; 36(21): 5129-5132, 2021 01 29.
Article in English | MEDLINE | ID: covidwho-1343669

ABSTRACT

MOTIVATION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused more than 14 million cases and more than half million deaths. Given the absence of implemented therapies, new analysis, diagnosis and therapeutics are of great importance. RESULTS: Analysis of SARS-CoV-2 genomes from the current outbreak reveals the presence of short persistent DNA/RNA sequences that are absent from the human genome and transcriptome (PmRAWs). For the PmRAWs with length 12, only four exist at the same location in all SARS-CoV-2. At the gene level, we found one PmRAW of size 13 at the Spike glycoprotein coding sequence. This protein is fundamental for binding in human ACE2 and further use as an entry receptor to invade target cells. Applying protein structural prediction, we localized this PmRAW at the surface of the Spike protein, providing a potential targeted vector for diagnostics and therapeutics. In addition, we show a new pattern of relative absent words (RAWs), characterized by the progressive increase of GC content (Guanine and Cytosine) according to the decrease of RAWs length, contrarily to the virus and host genome distributions. New analysis shows the same property during the Ebola virus outbreak. At a computational level, we improved the alignment-free method to identify pathogen-specific signatures in balance with GC measures and removed previous size limitations. AVAILABILITY AND IMPLEMENTATION: https://github.com/cobilab/eagle. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Humans , Protein Binding , SARS-CoV-2
6.
Brief Bioinform ; 22(2): 924-935, 2021 03 22.
Article in English | MEDLINE | ID: covidwho-1343628

ABSTRACT

In this paper, we present a toolset and related resources for rapid identification of viruses and microorganisms from short-read or long-read sequencing data. We present fastv as an ultra-fast tool to detect microbial sequences present in sequencing data, identify target microorganisms and visualize coverage of microbial genomes. This tool is based on the k-mer mapping and extension method. K-mer sets are generated by UniqueKMER, another tool provided in this toolset. UniqueKMER can generate complete sets of unique k-mers for each genome within a large set of viral or microbial genomes. For convenience, unique k-mers for microorganisms and common viruses that afflict humans have been generated and are provided with the tools. As a lightweight tool, fastv accepts FASTQ data as input and directly outputs the results in both HTML and JSON formats. Prior to the k-mer analysis, fastv automatically performs adapter trimming, quality pruning, base correction and other preprocessing to ensure the accuracy of k-mer analysis. Specifically, fastv provides built-in support for rapid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) identification and typing. Experimental results showed that fastv achieved 100% sensitivity and 100% specificity for detecting SARS-CoV-2 from sequencing data; and can distinguish SARS-CoV-2 from SARS, Middle East respiratory syndrome and other coronaviruses. This toolset is available at: https://github.com/OpenGene/fastv.


Subject(s)
SARS-CoV-2/isolation & purification , Sequence Analysis/methods , Viruses/isolation & purification , Algorithms , Genes, Viral , SARS-CoV-2/genetics , Viruses/genetics
7.
Emerg Infect Dis ; 27(8): 2052-2063, 2021 08.
Article in English | MEDLINE | ID: covidwho-1278367

ABSTRACT

Coronavirus disease has disproportionately affected persons in congregate settings and high-density workplaces. To determine more about the transmission patterns of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in these settings, we performed whole-genome sequencing and phylogenetic analysis on 319 (14.4%) samples from 2,222 SARS-CoV-2-positive persons associated with 8 outbreaks in Minnesota, USA, during March-June 2020. Sequencing indicated that virus spread in 3 long-term care facilities and 2 correctional facilities was associated with a single genetic sequence and that in a fourth long-term care facility, outbreak cases were associated with 2 distinct sequences. In contrast, cases associated with outbreaks in 2 meat-processing plants were associated with multiple SARS-CoV-2 sequences. These results suggest that a single introduction of SARS-CoV-2 into a facility can result in a widespread outbreak. Early identification and cohorting (segregating) of virus-positive persons in these settings, along with continued vigilance with infection prevention and control measures, is imperative.


Subject(s)
COVID-19 , SARS-CoV-2 , Disease Outbreaks , Humans , Minnesota/epidemiology , Phylogeny
9.
J Antimicrob Chemother ; 76(9): 2230-2233, 2021 08 12.
Article in English | MEDLINE | ID: covidwho-1276183

ABSTRACT

This article provides a brief overview of drug resistance to antiviral therapy as well as known and emergent variability in key SARS-CoV-2 viral sequences. The purpose is to stimulate deliberation about the need to consider drug resistance prior to widespread roll-out of antivirals for SARS-CoV-2. Many existing candidate agents have mechanisms of action involving drug targets likely to be critical for future drug development. Resistance emerged quickly with monotherapies deployed for other pulmonary viruses such as influenza virus, and in HIV mutations in key drug targets compromised efficacy of multiple drugs within a class. The potential for drug resistance in SARS-CoV-2 has not yet been rigorously debated or assessed, and we call for more academic and industry research on this potentially important future threat prior to widespread roll-out of monotherapies for COVID-19 treatment and prevention.


Subject(s)
COVID-19 , Coronavirus Infections , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Coronavirus Infections/drug therapy , Drug Resistance, Viral , Humans , SARS-CoV-2
10.
Acta Trop ; 221: 106013, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1272275

ABSTRACT

AIM: This study is looking for a common pathogenicity between SARS-CoV-2 and Plasmodium species, in individuals with certain HLA serotypes. METHODS: 1. Tblastx searches of SARS-CoV-2 are performed by limiting searches to five Plasmodium species that infect humans. 2. Aligned sequences in the respective organisms' proteomes are searched with blastp. 3. Binding predictions of the identified SARS-CoV-2 peptide to HLA supertype representatives are performed. 4. Blastp searches of predicted epitopes that bind strongly to the identified HLA allele are performed by limiting searches to H. sapiens and Plasmodium species, separately. 5. Peptides with minimum 60% identity to the predicted epitopes are found in results. 6. Peptides among those, which bind strongly to the same HLA allele, are predicted. 7. Step-4 is repeated by limiting searches to H. sapiens, followed by the remaining steps until step-7, for peptides sourced by Plasmodium species after step-6. RESULTS: SARS-CoV-2 peptide with single letter amino acid code CFLGYFCTCYFGLFC has the highest identity to P. vivax. Its YFCTCYFGLF part is predicted to bind strongly to HLA-A*24:02. Peptides in the human proteome both homologous to YFCTCYFGLF and with a strong binding affinity to HLA-A*24:02 are YYCARRFGLF, YYCHCPFGVF, and YYCQQYFFLF. Such peptides in the Plasmodium species' proteomes are FFYTFYFELF, YFVACLFILF, and YFPTITFHLF. The first one belonging to P. falciparum has a homologous peptide (YFYLFSLELF) in the human proteome, which also has a strong binding affinity to the same HLA allele. CONCLUSION: Immune responses to the identified-peptides with similar sequences and strong binding affinities to HLA-A*24:02 can be related to autoimmune response risk in individuals with HLA-A*24:02 serotypes, upon getting infected with SARS-CoV-2 or P. falciparum.


Subject(s)
COVID-19 , HLA-A24 Antigen , Malaria, Vivax , Peptides , Epitopes, T-Lymphocyte , Humans , Peptides/genetics , SARS-CoV-2
11.
Front Genet ; 12: 663098, 2021.
Article in English | MEDLINE | ID: covidwho-1268247

ABSTRACT

Symptoms of coronavirus disease 2019 (COVID-19) range from asymptomatic to severe pneumonia and death. A deep understanding of the variation of biological characteristics in severe COVID-19 patients is crucial for the detection of individuals at high risk of critical condition for the clinical management of the disease. Herein, by profiling the gene expression spectrum deduced from DNA coverage in regions surrounding transcriptional start site in plasma cell-free DNA (cfDNA) of COVID-19 patients, we deciphered the altered biological processes in the severe cases and demonstrated the feasibility of cfDNA in measuring the COVID-19 progression. The up- and downregulated genes in the plasma of severe patient were found to be closely related to the biological processes and functions affected by COVID-19 progression. More importantly, with the analysis of transcriptome data of blood cells and lung cells from control group and cases with severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection, we revealed that the upregulated genes were predominantly involved in the viral and antiviral activity in blood cells, reflecting the intense viral replication and the active reaction of immune system in the severe patients. Pathway analysis of downregulated genes in plasma DNA and lung cells also demonstrated the diminished adenosine triphosphate synthesis function in lung cells, which was evidenced to correlate with the severe COVID-19 symptoms, such as a cytokine storm and acute respiratory distress. Overall, this study revealed tissue involvement, provided insights into the mechanism of COVID-19 progression, and highlighted the utility of cfDNA as a noninvasive biomarker for disease severity inspections.

12.
Vaccine ; 39(30): 4108-4116, 2021 07 05.
Article in English | MEDLINE | ID: covidwho-1267950

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), initially originated in China in year 2019 and spread rapidly across the globe within 5 months, causing over 96 million cases of infection and over 2 million deaths. Huge efforts were undertaken to bring the COVID-19 vaccines in clinical development, so that it can be made available at the earliest, if found to be efficacious in the trials. We developed a candidate vaccine ZyCoV-D comprising of a DNA plasmid vector carrying the gene encoding the spike protein (S) of the SARS-CoV-2 virus. The S protein of the virus includes the receptor binding domain (RBD), responsible for binding to the human angiotensin converting enzyme (ACE-2) receptor. The DNA plasmid construct was transformed into E. coli cells for large scale production. The immunogenicity potential of the plasmid DNA has been evaluated in mice, guinea pig, and rabbit models by intradermal route at 25, 100 and 500 µg dose. Based on the animal studies proof-of-concept has been established and preclinical toxicology (PCT) studies were conducted in rat and rabbit model. Preliminary animal study demonstrates that the candidate DNA vaccine induces antibody response including neutralizing antibodies against SARS-CoV-2 and also elicited Th-1 response as evidenced by elevated IFN-γ levels.


Subject(s)
COVID-19 , Vaccines, DNA , Animals , Antibodies, Neutralizing , Antibodies, Viral , Antibody Formation , COVID-19 Vaccines , China , Escherichia coli , Guinea Pigs , Humans , Mice , Models, Animal , Rabbits , Rats , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics
13.
PLoS Negl Trop Dis ; 15(5): e0009374, 2021 05.
Article in English | MEDLINE | ID: covidwho-1264204

ABSTRACT

The development of efficient vaccines against COVID-19 is an emergent need for global public health. The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major target for the COVID-19 vaccine. To quickly respond to the outbreak of the SARS-CoV-2 pandemic, a nucleic acid-based vaccine is a novel option, beyond the traditional inactivated virus vaccine or recombinant protein vaccine. Here, we report a DNA vaccine containing the spike gene for delivery via electroporation. The spike genes of SARS-CoV and SARS-CoV-2 were codon optimized for mammalian cell expression and then cloned into mammalian cell expression vectors, called pSARS-S and pSARS2-S, respectively. Spike protein expression was confirmed by immunoblotting after transient expression in HEK293T cells. After immunization, sera were collected for antigen-specific antibody and neutralizing antibody titer analyses. We found that both pSARS-S and pSARS2-S immunization induced similar levels of antibodies against S2 of SARS-CoV-2. In contrast, only pSARS2-S immunization induced antibodies against the receptor-binding domain of SARS-CoV-2. We further found that pSARS2-S immunization, but not pSARS-S immunization, could induce very high titers of neutralizing antibodies against SARS-CoV-2. We further analyzed SARS-CoV-2 S protein-specific T cell responses and found that the immune responses were biased toward Th1. Importantly, pSARS2-S immunization in hamsters could induce protective immunity against SARS-CoV-2 challenge in vivo. These data suggest that DNA vaccination could be a promising approach for protecting against COVID-19.


Subject(s)
COVID-19/prevention & control , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccines, DNA/standards , Animals , Chlorocebus aethiops , Cricetinae , Electroporation , HEK293 Cells , Humans , Mesocricetus , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Plasmids , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Vaccines, DNA/immunology , Vero Cells
14.
Nucleic Acids Res ; 49(15): e90, 2021 09 07.
Article in English | MEDLINE | ID: covidwho-1262154

ABSTRACT

Variant visualization plays an important role in supporting the viral evolution analysis, extremely valuable during the COVID-19 pandemic. VirusViz is a web-based application for comparing variants of selected viral populations and their sub-populations; it is primarily focused on SARS-CoV-2 variants, although the tool also supports other viral species (SARS-CoV, MERS-CoV, Dengue, Ebola). As input, VirusViz imports results of queries extracting variants and metadata from the large database ViruSurf, which integrates information about most SARS-CoV-2 sequences publicly deposited worldwide. Moreover, VirusViz accepts sequences of new viral populations as multi-FASTA files plus corresponding metadata in CSV format; a bioinformatic pipeline builds a suitable input for VirusViz by extracting the nucleotide and amino acid variants. Pages of VirusViz provide metadata summarization, variant descriptions, and variant visualization with rich options for zooming, highlighting variants or regions of interest, and switching from nucleotides to amino acids; sequences can be grouped, groups can be comparatively analyzed. For SARS-CoV-2, we manually collect mutations with known or predicted levels of severity/virulence, as indicated in linked research articles; such critical mutations are reported when observed in sequences. The system includes light-weight project management for downloading, resuming, and merging data analysis sessions. VirusViz is freely available at http://gmql.eu/virusviz/.


Subject(s)
COVID-19/virology , Data Visualization , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Amino Acid Sequence , Base Sequence , Databases, Factual , Humans , Knowledge Bases , SARS-CoV-2/classification , South Africa/epidemiology , United States/epidemiology
15.
Virol J ; 18(1): 110, 2021 06 02.
Article in English | MEDLINE | ID: covidwho-1255943

ABSTRACT

BACKGROUND: The reliable detection of SARS-CoV-2 has become one of the most important contributions to COVID-19 crisis management. With the publication of the first sequences of SARS-CoV-2, several diagnostic PCR assays have been developed and published. In addition to in-house assays the market was flooded with numerous commercially available ready-to-use PCR kits, with both approaches showing alarming shortages in reagent supply. AIM: Here we present a resource-efficient in-house protocol for the PCR detection of SARS-CoV-2 RNA in patient specimens (RKI/ZBS1 SARS-CoV-2 protocol). METHODS: Two duplex one-step real-time RT-PCR assays are run simultaneously and provide information on two different SARS-CoV-2 genomic regions. Each one is duplexed with a control that either indicates potential PCR inhibition or proves the successful extraction of nucleic acid from the clinical specimen. RESULTS: Limit of RNA detection for both SARS-CoV-2 assays is below 10 genomes per reaction. The protocol enables testing specimens in duplicate across the two different SARS-CoV-2 PCR assays, saving reagents by increasing testing capacity. The protocol can be run on various PCR cyclers with several PCR master mix kits. CONCLUSION: The presented RKI/ZBS1 SARS-CoV-2 protocol represents a cost-effective alternative in times of shortages when commercially available ready-to-use kits may not be available or affordable.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Coronavirus Envelope Proteins/genetics , High-Throughput Nucleotide Sequencing/methods , Humans , Limit of Detection , Polyproteins/genetics , RNA, Viral/genetics , SARS-CoV-2/isolation & purification , Sensitivity and Specificity , Viral Proteins/genetics
16.
PLoS One ; 16(5): e0251368, 2021.
Article in English | MEDLINE | ID: covidwho-1242246

ABSTRACT

COVID-19 is challenging healthcare preparedness, world economies, and livelihoods. The infection and death rates associated with this pandemic are strikingly variable in different countries. To elucidate this discrepancy, we analyzed 2431 early spread SARS-CoV-2 sequences from GISAID. We estimated continental-wise admixture proportions, assessed haplotype block estimation, and tested for the presence or absence of strains' recombination. Herein, we identified 1010 unique missense mutations and seven different SARS-CoV-2 clusters. In samples from Asia, a small haplotype block was identified, whereas samples from Europe and North America harbored large and different haplotype blocks with nonsynonymous variants. Variant frequency and linkage disequilibrium varied among continents, especially in North America. Recombination between different strains was only observed in North American and European sequences. In addition, we structurally modelled the two most common mutations, Spike_D614G and Nsp12_P314L, which suggested that these linked mutations may enhance viral entry and replication, respectively. Overall, we propose that genomic recombination between different strains may contribute to SARS-CoV-2 virulence and COVID-19 severity and may present additional challenges for current treatment regimens and countermeasures. Furthermore, our study provides a possible explanation for the substantial second wave of COVID-19 presented with higher infection and death rates in many countries.


Subject(s)
Recombination, Genetic , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Virulence/physiology , COVID-19/pathology , COVID-19/virology , Databases, Genetic , Genetic Variation , Haplotypes , Humans , Linkage Disequilibrium , Molecular Dynamics Simulation , Mutation, Missense , Principal Component Analysis , Protein Structure, Tertiary , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Severity of Illness Index , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
17.
J Virol Methods ; 295: 114197, 2021 09.
Article in English | MEDLINE | ID: covidwho-1240483

ABSTRACT

OBJECTIVES: The SARS-CoV-2 pandemic has created an unprecedented need for rapid large-scale diagnostic testing to prompt clinical and public health interventions. Currently, several quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assays recommended by the World Health Organization are being used by clinical and public health laboratories and typically target regions of the RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N) coding region. However, it is currently unclear if results from different tests are comparable. This study aimed to clarify the clinical performances of the primer/probe sets designed by US CDC and Charité/Berlin to help clinical laboratories in assay selection for SARS-CoV-2 routine detection. METHODS: We compared the clinical performances of the recommended primer/probe sets using one hundred nasopharyngeal swab specimens from patients who were clinically diagnosed with COVID-19. An additional 30 "pre-intervention screening" samples from patients who were not suspected of COVID-19 were also included in this study. We also performed sequence alignment between 31064 European SARS-CoV-2 and variants of concern genomes and the recommended primer/probe sets. RESULTS: The present study demonstrates substantial differences in SARS-CoV-2 RNA detection sensitivity among the primer/probe sets recommended by the World Health Organization especially for low-level viral loads. The alignment of thousands of SARS-CoV-2 sequences reveals that the genetic diversity remains relatively low at the primer/probe binding sites. However, multiple nucleotide mismatches might contribute to false negatives. CONCLUSION: An understanding of the limitations depending on the targeted genes and primer/probe sets may influence the selection of molecular detection assays by clinical laboratories.


Subject(s)
DNA Primers/genetics , Genome, Viral/genetics , SARS-CoV-2/isolation & purification , COVID-19/diagnosis , COVID-19/virology , COVID-19 Nucleic Acid Testing , Coronavirus/genetics , Humans , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/genetics , Sensitivity and Specificity , Sequence Alignment , Viral Load , Viral Proteins/genetics
18.
Proc Natl Acad Sci U S A ; 118(23)2021 06 08.
Article in English | MEDLINE | ID: covidwho-1238060

ABSTRACT

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic and has claimed over 2 million lives worldwide. Although the genetic sequences of SARS-CoV and SARS-CoV-2 have high homology, the clinical and pathological characteristics of COVID-19 differ significantly from those of SARS. How and whether SARS-CoV-2 evades (cellular) immune surveillance requires further elucidation. In this study, we show that SARS-CoV-2 infection leads to major histocompability complex class Ι (MHC-Ι) down-regulation both in vitro and in vivo. The viral protein encoded by open reading frame 8 (ORF8) of SARS-CoV-2, which shares the least homology with SARS-CoV among all viral proteins, directly interacts with MHC-Ι molecules and mediates their down-regulation. In ORF8-expressing cells, MHC-Ι molecules are selectively targeted for lysosomal degradation via autophagy. Thus, SARS-CoV-2-infected cells are much less sensitive to lysis by cytotoxic T lymphocytes. Because ORF8 protein impairs the antigen presentation system, inhibition of ORF8 could be a strategy to improve immune surveillance.


Subject(s)
Antigen Presentation , COVID-19/immunology , Down-Regulation/immunology , Histocompatibility Antigens Class I/immunology , Immune Evasion , SARS-CoV-2/immunology , Viral Proteins/immunology , Animals , Autophagy/genetics , Autophagy/immunology , COVID-19/genetics , Chlorocebus aethiops , HEK293 Cells , Histocompatibility Antigens Class I/genetics , Humans , Lysosomes/genetics , Lysosomes/immunology , Lysosomes/virology , Mice , Mice, Transgenic , SARS-CoV-2/genetics , Vero Cells , Viral Proteins/genetics
19.
Comput Biol Med ; 134: 104451, 2021 07.
Article in English | MEDLINE | ID: covidwho-1237662

ABSTRACT

COVID-19, a global pandemic caused by an RNA virus named SARS-CoV-2 has brought the world to a standstill in terms of infectivity, casualty, and commercial plummet. RNA viruses can encode microRNAs (miRNAs) capable of modulating host gene expression, and with that notion, we aimed to predict viral miRNA like sequences of MERS-CoV, SARS-CoV and SARS-CoV-2, analyze sequence reciprocity and investigate SARS-CoV-2 encoded potential miRNA-human genes interaction using bioinformatics tools. In this study, we retrieved 206 SARS-CoV-2 genomes, executed phylogenetic analysis, and the selected reference genome (MT434792.1) exhibited about 99% similarities among the retrieved genomes. We predicted 402, 137, and 85 putative miRNAs of MERS-CoV (NC_019843.3), SARS-CoV (NC_004718.3), and SARS-CoV-2 (MT434792.1) genome, respectively. Sequence similarity was analyzed among 624 miRNAs which revealed that the predicted miRNAs of SARS-CoV-2 share a cluster with the clad of miRNAs from MERS-CoV and SARS-CoV. Only SARS-CoV-2 derived 85 miRNAs were encountered for target prediction and 29 viral miRNAs seemed to target 119 human genes. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis suggested the involvement of respective genes in various pathways and biological processes. Finally, we focused on eight putative miRNAs influencing 14 genes that are involved in the adaptive hypoxic response, neuroinvasion and hormonal regulation, and tumorigenic progression in patients with COVID-19. SARS-CoV-2 encoded miRNAs may cause misexpression of some critical regulators and facilitate viral neuroinvasion, altered hormonal axis, and tumorigenic events in the human host. However, these propositions need validation from future studies.


Subject(s)
COVID-19 , MicroRNAs , Computer Simulation , Humans , MicroRNAs/genetics , Phylogeny , SARS-CoV-2
20.
mSphere ; 6(3)2021 05 19.
Article in English | MEDLINE | ID: covidwho-1236422

ABSTRACT

Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can often generate spurious amplicons, especially in the absence of target sequences, resulting in false-positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets and can accumulate mutations that will lead to inefficient primer binding and thus false-negative results. Multiplex assays targeting different regions of the analyte and logical signal readout using sequence-specific probes can help to reduce both false negatives and false positives. Here, we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on a non-sequence-specific readout into individual and multiplex one-pot assays that can be visually read using sequence-specific oligonucleotide strand exchange (OSD) probes. We describe both fluorescence-based and Boolean logic-gated colorimetric lateral flow readout methods and demonstrate detection of SARS-CoV-2 virions in crude human saliva.IMPORTANCE One of the key approaches to treatment and control of infectious diseases, such as COVID-19, is accurate and rapid diagnostics that is widely deployable in a timely and scalable manner. To achieve this, it is essential to go beyond the traditional gold standard of quantitative PCR (qPCR) that is often faced with difficulties in scaling due to the complexity of infrastructure and human resource requirements. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), have been long pursued as ideal, low-tech alternatives for rapid, portable testing. However, isothermal approaches often suffer from false signals due to employment of nonspecific readout methods. We describe general principles for rapidly converting nonspecifically read LAMP assays into assays that are read in a sequence-specific manner by using oligonucleotide strand displacement (OSD) probes. We also demonstrate that inclusion of OSD probes in LAMP assays maintains the simplicity of one-pot assays and a visual yes/no readout by using fluorescence or colorimetric lateral-flow dipsticks while providing accurate sequence-specific readout and the ability to logically query multiplex amplicons for redundancy or copresence. These principles not only yielded high-surety isothermal assays for SARS-CoV-2 but might also aid in the design of more sophisticated molecular assays for other analytes.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , SARS-CoV-2/genetics , Saliva/virology , Humans , Point-of-Care Testing , RNA, Viral/genetics , SARS-CoV-2/isolation & purification
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